Methods for selective targeting

ABSTRACT

A selective targeting method is disclosed comprising contacting a library of ligands, particularly a peptide library, with an anti-target to allow the ligands to bind to the anti-target; separating the non-binding ligands from the anti-target bound ligands, contacting the non-binding anti-target ligands with a target allowing the unbound ligands to bind with the target to form a target-bound ligand complex; separating the target-bound ligand complex from ligands which do not bind to the target, and identifying the target-bound ligands on the target-bound ligand complex wherein the target-bound ligands have a K D  in the range of about 10 −7  to 10 −10  M. Additionally claimed are the ligands identified according to the method.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.12/099,632, filed Apr. 8, 2008, now U.S. Pat. No. 8,318,640, which is acontinuation of U.S. application Ser. No. 10/968,732, filed Oct. 19,2004, now abandoned, which is a continuation of U.S. application Ser.No. 09/832,723, filed Apr. 11, 2001, now abandoned, which claims benefitof and priority to U.S. Ser. No. 60/197, 259, filed Apr. 14, 2000, eachof which are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

The present invention is directed to methods for the selection andidentification of compounds capable of binding specifically to a targetin the presence of undesired background targets (anti-targets) usinglibraries of similar compounds. In one particular aspect, the presentinvention is related to the selection of ligands from peptide libraries.Ligand peptides identified according to the method of the invention havea binding affinity and a selectivity to a target similar to the bindingaffinity and selectivity of antibodies.

The literature is replete with examples of recent advances in methodsfor screening large library pools of compounds, especially peptides.Methods for screening these compounds to identify molecules that bind toa preselected target have also been advanced. One well-known method isbiopanning which was originally developed by Smith, G. P., (1985),Science 228:1315. Biopanning in its simplest form is an in vitroselection process in which a library of phage-displayed peptides isincubated with a target. The target and phage are allowed to bind andunbound phage are washed away. The specifically bound phage are thenacid eluted. The eluted pool of phage is amplified in vivo and theprocess is repeated. After a number of rounds individual clones areisolated and sequenced.

A number of variations of the biopanning technique first introduced bySmith have been described and reference is made to Christian et al.,(1992) J. Mol. Biol., 227:711; Cwirla et al., (1990) Proc. Natl. Acad.Sci. USA, 87:6378; Cull et al., (1992) Proc. Natl. Acad. Sci. USA,89:1865; Huls et al., (1996) Nature Biotechnol., 7:276; and Bartoli etal., (1998) Nature Biotechnol., 16:1068.

Huls et al., 1996 supra, describe a method comprising flowcytometry-based subtractive selection of phage antibody on intact tumorcells. The phage-displayed antibodies remain bound to the target duringthe flow-cytometric selection. However, prior to amplification thecell-bound phages are eluted from the target. WO 98/54312 disclosesselection of antibodies under mild conditions with high affinities forantigens using antibody libraries displayed on ribosomes.

In many prior art methods it is generally assumed that elution of targetbound ligands is sufficient to identify the tightest binding ligands ina library. However, a number of research papers report on low affinitybinders using elution techniques (U.S. Pat. No. 5,582,981).Nevertheless, physical separation of the ligands from the target priorto amplification or identification is the standard method for selectingligands that bind to a preselected target.

Balass et al., (1996) Anal. Biochem., 243:264, describe the selection ofhigh-affinity phage-peptides from phage-peptide libraries using abiotinylated target immobilized on a nitrostreptavidin matrix. Theinteracting phage particles were released under conventional acidelution. Further, after acid elution, the target complex was analyzedfor bound phage. These particles were exposed to alkaline solutions orfree biotin to release the target bound phage particles from the solidsupport. The affinity of the isolated phage was found to be higher thanthe phage released by traditional acid elution methods. However, thesynthetically prepared peptides exhibited a lower affinity for thetarget than the peptides prepared from sequences obtained by acid-elutedphage.

Other targeting methods include, for example, SELEX. This is a procedurein which an oligonucleotide from a library of randomized sequences isembedded in a pool of nucleic acids. Many cycles of affinity selectionto a target of the oligonucleotide from the heterologous RNA or DNApopulation occurs. The target and annealed nucleic acids are partitionedand amplified. In order to proceed to the amplification step, selectednucleic acids must be released from the target after partitioning. (U.S.Pat. No. 5,475,096)

While various methods for screening and selecting libraries of compoundsexist, improved methods that do not require multiple rounds of selectionare particularly needed for compounds that a) bind tightly andspecifically to targets that are not well-defined at the chemical,biochemical or genetic level but have macroscopic properties that aredesirable to target, b) bind tightly and specifically to targets thatcannot be easily physically separated from a large background ofundesirable targets (anti-targets), and c) bind to targets under harshconditions, such as acidic pH, high detergent concentration or hightemperature.

The selective targeting method according to the invention overcomes someof the above deficiencies of the prior art methods and in particularoffers an advantage in rapidly identifying compounds, particularlypeptides, that bind with a high affinity and selectively to a target.

SUMMARY OF THE INVENTION

In one aspect, the invention concerns a method for screening a ligandlibrary comprising contacting the ligand library with an anti-target toallow the ligands to bind with the anti-target; separating unboundligands and contacting said unbound ligands with the selected target toallow said unbound ligands to bind with the target to form atarget-bound ligand complex; separating said target-bound ligand complexfrom ligands which do not bind to said target; and identifying thetarget-bound ligands on the target-bound ligand complex.

In another aspect, the invention concerns a method for screening aligand library comprising contacting the ligand library essentiallysimultaneously with a selected target and an anti-target to allow theligands to bind with the target forming a target-bound ligand complex;separating the target-bound ligand complex from the anti-target,anti-target bound ligands and free ligands; and identifying the ligandsof the target-bound ligand complex. The contacting step may beaccomplished either in vivo or in vitro.

In one preferred embodiment, the selectivity of ligand binding to atarget compared to ligand binding to an anti-target is about at least10:1. In a second preferred embodiment, the ligand is a peptide but notan antibody and is bound to the target with a K_(D) at least about 10⁻⁷M and preferably in the range of about 10⁻⁷ M to 10⁻¹⁰ M. In a thirdpreferred embodiment, the ligand library is a peptide library.Preferably the peptides identified according to the method are less than25 amino acids in length and more preferably between 4 to 15 amino acidsin length. In a fourth embodiment, the k_(off) is about 10⁻⁴ sec⁻¹ orless. In a fifth embodiment, the target is a stain, and particularly astain on fabric, wherein the stain is a porphyrin derived stain, atannin derived stain, a carotenoid pigment derived stain, an anthocyaninpigment derived stain, a soil-based stain, oil-based stain, or humanbody soil stains.

In yet a further aspect, the invention is directed to the ligands,particularly peptide ligands, which are identified by the selectivetargeting method of the invention.

Another embodiment of the invention concerns a method for identifyingpeptides useful in a cleaning composition comprising, contacting apeptide library with an anti-target to allow the peptides to bind withthe anti-target, wherein the anti-target is selected from the groupconsisting of fabric, ceramic, glass, stainless steel, and plastic;separating unbound anti-target peptides, contacting the unboundanti-target peptides with a target wherein the target is a stainselected from the group consisting of porphyrin derived stains, tanninderived stains, carotenoid pigment derived stains, anthocyanin pigmentderived stains, soil-based derived stains, oil-based derived stains andhuman body soil stains to allow the unbound peptides to bind with thestain to form a stain-bound peptide complex; and identifying thestain-bound peptide on the stain-bound peptide complex. In at least oneembodiment the peptide binds to the stain with a K_(D) in the range ofabout 10⁻⁷M to 10⁻¹⁰M.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a general schematic diagram of the selective targeting methoddisclosed herein. The method comprises the steps of, a) selectionagainst anti-targets which provides a library of ligands depleted ofanti-target bound ligands, b) selection for the target by formation of atarget-bound ligand complex, c) separation of the target-bound ligandcomplex, d) identification of the target-bound ligands, and e)optionally sequencing the target-bound ligands, exposing thetarget-bound ligands to additional rounds of selective targeting, and/ordiversification.

FIG. 2 is a photograph of a gel of PCR amplified DNA fragments afterlysis of TNF-α bound phage (third lane), IL-6 bound phage (fourth lane),and IL-8 bound phage (fifth lane). The first lane is the DNA marker andthe second lane is a phage clone control.

FIGS. 3A and 3B are photographs of gels of PCR amplified DNA fragmentsfor control (FIG. 3A) and soil-targeted peptides (FIG. 3B).

FIG. 4A illustrates binding, dissociation and attempted elution of phagepeptide clone A1 corresponding to RYWQDIP (SEQ ID NO: 3) fromimmobilized TNF-α on an IAsys biosensor cuvette. FIG. 4B is a cartoonillustrating the process of identifying tight binding phage.

FIGS. 5A and 5B are images of collar soils and the correspondingpolyester fabric as viewed by digital imaging and autoradiography,respectively.

FIGS. 6A and 6B illustrate the fractional percent ¹⁴C labeled peptidebinding to collar soils on polyester cotton fabric. FIG. 6A illustratesa soil-targeted peptide, SISSTPRSYHWT, (SEQ ID NO: 20) which isterminally labeled with ¹⁴C-glycine wherein ∘ depicts stain #1, ▪depicts stain #2, and ∀ depicts blue polycotton and FIG. 6B illustratesa random peptide, NFFPTWILPEHT (SEQ ID NO: 78) which is terminallylabeled with ¹⁴C-glycine.

FIG. 7 illustrates the kinetics of dissociation of the Ni-chelatedpeptide GGHTFQHQWTHQTR (SEQ ID NO: 28) from collar soil (•) and thecorresponding cotton fabric (∘). The slope of the lines correspond torate constants k_(off)=1×10⁻³ sec⁻¹.

FIG. 8 is a photograph of a gel of PCR amplified fragment for egg soiltargets and stainless steel or glass bead anti-targets.

FIG. 9 illustrates ELISA assay results for binding of 3 peptides.LESTPKMK (SEQ ID NO: 115) binds to hair and FTQSLPR (SEQ ID NO: 116)selectively targets skin and not hair (▪ depicts hair and ∀ depictsskin).

DETAILED DESCRIPTION OF THE INVENTION

A. Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. For the purposes ofthe present invention, the following terms are used to describe theinvention herein.

The term “ligand” refers to a molecule or compound that is recognized bya particular target or anti-target. The term is independent of molecularsize or compositional feature. The ligand may serve as a substrate foran enzyme-catalyzed reaction, as an agonist, as an antagonist, act as asignal messenger, or stimulate or inhibit metabolic pathways. Ligandsmay be nucleic acids, peptides, peptide derivatives, peptidomimetics,polypeptides, small organic molecules, carbohydrates and other moleculesthat are isolated from a candidate mixture that acts on a target in adesirable manner. Preferably the desirable manner is binding the target,but could include for example, catalytically changing the target orreacting with the target that modifies or alters the target. In onepreferred embodiment, the ligand has a binding affinity for the targetin the range of an antibody binding affinity for a selected receptor.

The term “library” refers to a collection of chemical or biologicalentities that can be created in a single reservoir and simultaneouslyscreened for a desired property. As used herein a library can have aminimum size of at least two members and may contain as many as 10¹⁵members. In one aspect, the library has at least 10² members. In anotheraspect, the library has at least 10³ members. In yet another aspect, thelibrary has at least 10⁶ members. In a further aspect, the library hasat least 10⁹ members. The size of a library refers to the total numberof entities comprising the library whether the members are the same ordifferent.

A “peptide library” refers to a set of peptides and to the peptides andany fusion proteins containing those peptides. Stochastic or randomprocesses may be used to construct random peptides. The term “random”does not mean that the library composition is not known.

The term “peptide” refers to an oligomer in which the monomeric unitsare amino acids (typically, but not limited to L-amino acids) linked byan amide bond. Peptides may be two or more amino acids in length.Peptides identified according to the invention are preferably less than50 amino acids in length, more preferably less than 30 amino acids inlength, also preferably less than 25 amino acids in length, andpreferably less than 20 amino acids in length. In one preferredembodiment the peptides identified according to the method of theinvention are between 4 and 15 amino acids in length. However, ingeneral peptides may be up to 100 amino acids in length. Peptides thatare longer than 100 amino acids in length are generally referred to aspolypeptides. Standard abbreviations for amino acids are used herein.(See Singleton et al., (1987) Dictionary of Microbiology and MolecularBiology, Second Ed., page 35, incorporated herein by reference).

The peptides or polypeptides may be provided as a fusion peptide orprotein. Peptides include synthetic peptide analogs wherein the aminoacid sequence is known. The term peptide does not include moleculesstructurally related to peptides, such as peptide derivatives orpeptidomimetics whose structure cannot be determined by standardsequencing methodologies, but rather must be determined by more complexmethodologies such as mass spectrometric methods. Peptidomimetics (alsoknown as peptide mimetics) are peptide analogs but are non-peptidecompounds. Usually one or more peptide linkages are optionally replaced.(Evans et al., (1987) J. Med. Chem. 30:1229). The term “protein” is wellknown and refers to a large polypeptide.

The term “nucleic acid” means DNA, RNA, single-stranded ordouble-stranded and chemical modifications thereof. Modifications mayinclude but are not limited to modified bases, backbone modifications,methylations, unusual base pairing modifications, and cappingmodifications. When a nucleic acid library is used in the selectivetargeting method of the invention, the nucleic acid ligand is generallybetween 4 and 250 nucleotides in length, and preferably between 4 and 60nucleotides in length.

The invention further includes ligands, preferably nucleic acid, peptideor polypeptide ligands and more preferably peptide ligands that havesubstantially the same ability to bind to a target as the nucleic acid,peptide or polypeptide identified by the selective targeting methoddescribed herein. Substantially the same ability to bind a target meansthe affinity and selectivity is approximately the same as the affinityand selectivity of the ligands selected by the method herein claimed.

Additionally a ligand having substantially the same ability to bind to atarget will be substantially homologous to the ligand identified by thedisclosed selective targeting method. With respect to a nucleic acidsequence, substantially homologous to an identified ligand means thedegree of primary sequence homology is in excess of 80%, preferably inexcess of 85%, more preferably in excess of 90%, further preferably inexcess of 95%, even more preferably in excess of 97%, and mostpreferably in excess of 99%. It will be appreciated by those skilled inthe art that as a result of the degeneracy of the genetic code, amultitude of peptide encoding nucleotide sequences may be produced. Apeptide or polypeptide is substantially homologous to a referencepeptide or polypeptide if it has at least 85% sequence identity,preferably at least 90% to 95% sequence identity, more preferably atleast 97%, and most preferably at least 99% identical or equivalent tothe reference sequence when optimally aligned. Optimal alignment of thesequences may be conducted by various known methods and computerizedimplementation of known algorithims (e.g. TFASTA, BESTFIT, in theWisconsin Genetics Software Package, Release 7.0, Genetics ComputerGroup, Madison, Wis.). General categories of equivalent amino acidsinclude 1) glutamic acid and aspartic acid; 2) lysine, arginine, andhistidine; 3) alanine, valine, leucine, and isoleucine; 4) asparagineand glutamine; 5) threonine and serine; 6) phenylalanine, tyrosine andtryptophan; and 7) glycine and alanine. It is well within the ordinaryskill of those in the art to determine whether a given sequencesubstantially homologous to those identified herein have substantiallythe same ability to bind a target.

A small organic molecule as defined herein is a molecule, preferably anonpolymeric molecule, having a molecular weight of approximately 1000daltons or less and more preferably 500 daltons or less. A “peptoid” isdefined herein as an enzymatically resistant peptide analog.

The term “target” or “anti-target” refers to molecules or heterogeneousmolecules that have a binding affinity as defined herein, for a givenligand. Both target and anti-targets may be naturally occurring orsynthetic molecules or heterogeneous molecules.

The binding affinity of a ligand for its target or anti-target may bedescribed by the dissociation constant (K_(D)), concentration needed for50% effective binding (EC₅₀), or concentration needed for 50% inhibitionof binding of another compound that binds to the target (IC₅₀). K_(D) isdefined by k_(off)/k_(on). The k_(off) value defines the rate at whichthe target-ligand complex breaks apart or separates. This term issometimes referred to in the art as the kinetic stability of thetarget-ligand complex or the ratio of any other measurable quantity thatreflects the ratio of binding affinities, such as an enzyme-linkedimmunosorbent assay (ELISA) signal or radio-active label signal.Selectivity is defined by the ratio of binding affinities or k_(off) fordissociation of the ligand-complex (target K_(D) anti-target K_(D)). Thek_(on) value describes the rate at which the target and ligand combineto form the target-ligand complex.

The term “contacting” is broadly defined to mean placing a library ofligands and a target or anti-target in immediate proximity orassociation and includes in vitro and in vivo contact. The term includestouching, associating, joining, combining, intravenous injection, oraladministration, intraperitoneally, topical application, intramuscular,inhalation, subcutaneous application and the like. The term “separating”as used herein means to select, segregate, partition, isolate, collect,keep apart and disunite.

“Amplifying” means a process or combination of process steps thatincreases the amount or number of copies of a molecule or class ofmolecules. In one aspect, amplification refers to the production ofadditional copies of nucleic acid sequences that is carried out usingpolymerase chain reaction (PCR) technology well known in the art. Inanother aspect, amplification refers to production of phage virions byinfection of a host.

As used in the specification and claims, the singular “a”, “an” and“the” include the plural references unless the context clearly dictatesotherwise. For example, the term “a protease” may include a plurality ofproteases.

The following references describe the general techniques employedherein: Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Innis etal., PCR Protocols—A Guide to Methods and Applications (1990), AcademicPress, Inc.; Kay et al., (1996) Phage Display of Peptides and Proteins,Academic Press; Ausubel et al., (1987) Current Protocols in MolecularBiology, Greene-Publishing & Wiley Interscience NY (Supplemented through1999); Berger and Kimmel, (1987) Methods in Enzymology, Vol. 152.Academic Press Inc., San Diego, Calif.

The contents of all references, patents and published patentapplications cited throughout this application are hereby incorporatedby reference in their entirety.

B. General Method

Described herein is a selective targeting method for screening a libraryof ligands having a binding affinity and selectivity for a selectedtarget. In its most basic form the selective targeting method may bedefined as follows: Preparing or obtaining a library of ligands,preferably peptides of different sequences and more preferably a randompeptide library. Deselecting ligands that bind with an anti-target bycontacting the ligand library with an anti-target under conditionsfavorable for binding between the ligands of the library and theanti-target; allowing the anti-target to bind with the ligands; andseparating the anti-target non-binders (unbound ligands) from theanti-target ligand bound molecules and any free ligands. Contacting theanti-target non-binders with a selected target under suitable conditionsand allowing them to bind. Ligands with an affinity for the target willbind to form a target-bound ligand complex. The removal of ligands boundto the anti-target and removal of weak target-bound ligands maygenerally be referred to as library depletion. The target-bound ligandcomplex is then separated from the remaining mixture including theunbound ligands, and the target-bound ligands are identified. Thetarget-bound ligand complex or the target-bound ligands may thenoptionally be subjected to amplification, sequencing or further roundsof selection (FIG. 1). The invention further comprises the ligandsidentified according to the selective targeting method of the invention.

In the practice of the invention, a library of compounds to be testedwill generally be provided. A library of ligands may include, but is notlimited to, random peptide libraries, synthetic peptide orpeptidomimetic combinatorial libraries, peptide loop libraries,combinatorial chemical libraries, and oligonucleotide libraries. Theselibraries are well known to those in the art as well as methods formaking said libraries. Reference is made to Barbas, C. F. (1993) CurrentOpinion in Biotech., 4:526; Cwirla et al., (1990) supra; Scott andSmith, (1990) Science, 249:386; Cull et al., (1992) supra; Pinilla etal., (1994) Biochem. J. 301:847; Sambrook et al., (1989) supra; Ausubelet al., (1987) supra; and Gubler and Hoffman, (1983) Gene 25:263; eachof which is incorporated herein by reference.

One preferred type of library includes random peptide libraries (alsosometimes referred to in the art as epitope libraries). These librariesmay include cell-surface display libraries, for example yeast display(Boder and Wittrup (1997) Nat. Biotechnol., 15:553); peptide librariesinserted into proteins (Lenstra et al., (1992) J. Immunol. Methods,152:149 and U.S. Pat. No. 5,837,500); direct screening of peptides onpolysomes (Tuerk et al., (1990) Science 249:505) and phage displaylibraries (Delvin et al., (1990) Science 249:404; WO91/18980; Dower etal. WO91/19818; and Parmley et al., (1988) Gene 73:305). Phage displaylibraries are particularly preferred. A phage display library is alibrary in which numerous peptides are displayed on the surface of abacteriophage, such as a filamentous phage. The peptide or protein isexpressed as a fusion with a coat protein of the bacteriophage resultingin display of the fusion protein on the surface of the virion while theDNA encoding the fusion resides within the virion. Suitable non-limitingexamples of vectors for construction of phage libraries include fAFF1;the fUSE series, such as fUSE5; lamba phage vectors; and T7select(non-filamentous) phage vectors. (Smith and Scott (1993) MethodsEnzymol. 217:228; and Cwirla et al., (1990) Proc. Natl. Acad. Sci. USA87:6378). Phage-peptide library kits are available and reference is madeto Chiron Corp. (Emeryville, Calif.), New England BioLabs Inc., CatalogNo. 8100 (Beverly, Mass.), and Novagen Catalog No. 70550-3 (MadisonWis.). While various antibody libraries are known, including antibodydisplay libraries on phage (de Bruin et al., (1999) Nat. Biotechnol.,17:397), in one preferred aspect of the present invention, the libraryof ligands used in the selective targeting method according to theinvention will not include antibodies.

Another type of peptide library encoded by nucleic acids includes alibrary wherein the peptide is expressed as a fusion with anotherprotein, for example, either a cell-surface protein or an internalprotein of a host. The nucleotides encoding the peptide are insertedinto a gene encoding the internal protein. Various examples of this typeof library include the fusion of peptides to a lac repressor, GAL4,thioredoxin, and various antibodies (U.S. Pat. Nos. 5,283,173;5,270,181; and 5,292,646). Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865 teach the construction of a fusion gene encoding a fusionprotein of peptide library members and Lacl. Nucleic acids encoding alibrary of peptides are inserted into a gene encoding Lacl. The fusionprotein and the fusion plasmid encoding the fusion protein arephysically linked by binding of the peptides to the lac operatorsequence in a plasmid. Host cells may be transformed with the libraryplasmids. The cells expressing the fusion protein are lysed releasingthe fusion protein and associated DNA (see for example U.S. Pat. No.5,733,731). The library can then be screened or selected. DNA shuffledlibraries are also known which are constructed by homologous exchange ofDNA fragments during DNA recombination methods or by synthetic methods(see for example U.S. Pat. No. 5,605,793 and Stemmer (1994), Proc. Natl.Aca. Sci. USA 91:10747).

So called anchor libraries have been described in PCT US96/09383 and WO97/22617. This is a peptide library wherein peptides have non-continuousregions of random amino acids separated by specifically designated aminoacids. These libraries are made by genetic or chemical means.

A combinatorial chemical library and particularly a peptide library mayalso be synthesized directly by methods known in the art including, butnot limited to synthesis by arrays (Foder et al., (1991) Science251:767); synthesis on solid supports (WO97/35198); and other chemicalmethods such as those disclosed in Lam et al., (1993) Bioorg. Med. Chem.Lett., 3:419, Tjoeng et al., (1990) Int. J. Pept. Protein Res. 35:141,and WO96/33010.

Methods for creating combinatorial chemical libraries are also known inthe art. Combinatorial libraries include large numbers of chemicalvariants for peptides, oligonucleotides, peptoids, carbohydrates, smallorganic molecules and even solid-state materials (Schultz et al., (1995)Science, 268:1738). A core structure will be varied by addingsubstituents or by linking different molecular building blocks.Libraries may include molecules free in solution, linked to solidparticles or beads, or arrayed on surfaces of modified organisms.Virtually any class of compounds may be modified by varying substituentsaround the core molecule. Various non-limiting examples of classes ofcompounds for combinatorial libraries include benzodiazepines;mercaptoacyl prolines; carbamates; chalcone libraries; ketoamideconjugates; polyketones; paclitaxel libraries; anilides;aryloxyphenoxypropionates; oxazolidinones; carbohydrates; and numerousother classes. While methods for making combinatorial libraries are welldocumented in the literature, these methods may be very time consuming.Various companies now make instrumentation to generate combinatoriallibraries from both solution and solid phase synthesis (CombiChem. Inc.(San Diego, Calif.); Advanced ChemTech (Louisville); Zymark Corp. (MA);and Hewlett Packard (CA)). Once a library has been generated it canoptionally be purified for example by high performance liquidchromatography (HPLC). Once a small organic molecule is screened andidentified according to the selective targeting method of the invention,it may be produced on a larger scale by means of organic synthesis knownin the art.

As taught herein not only are standard methods for generating librariesof ligands well known, but also ligand libraries may be obtainedcommercially, for example from Sigma (St. Louis Mo.) or from variouspublic sources such as American Type Culture Collection (ATCC) and theNational Institute of Health (NIH).

Suitable targets and anti-targets used in the selective targeting methodaccording to the invention include, but are not limited to, proteins,peptides, nucleic acids, carbohydrates, lipids, polysaccharides,glycoproteins, hormones, receptors, antigens, antibodies, viruses,pathogens, toxic substances, metabolites, inhibitors, drugs, dyes,nutrients, growth factors, cells or tissues.

Sources of cells or tissues include human, animal, bacterial, fungal,viral and plant. Tissues are complex targets and refer to a single celltype, a collection of cell types or an aggregate of cells generally of aparticular kind. Tissue may be intact or modified. General classes oftissue in humans include but are not limited to epithelial, connectivetissue, nerve tissue, and muscle tissue.

Preferred human cellular targets or anti-targets include hematopoieticcells, cancer cells and retroviral-mediated transduced cells.Hematopoietic cells encompass hematopoietic stem cells, erythrocytes,neutrophils, monocytes, platelets, mast cells, eosinophils, basophils, Band T cells, macrophages, and natural killer cells.

Non-limiting examples of protein and chemical targets encompassed by theinvention include chemokines and cytokines and their receptors.Cytokines as used herein refer to any one of the numerous factors thatexert a variety of effects on cells, for example inducing growth orproliferation. Non-limiting examples include interleukins (IL), IL-2,IL-3, IL-4 IL-6, IL-10, IL-12, IL-13, IL-14 and IL-16; soluble IL-2receptor; soluble IL-6 receptor; erythropoietin (EPO); thrombopoietin(TPO); granulocyte macrophage colony stimulating factor (GM-CSF); stemcell factor (SCF); leukemia inhibitory factor (LIF); interferons;oncostatin M(OM); the immunoglobulin superfamily; tumor necrosis factor(TNF) family, particularly TNF-α; TGFβ; and IL-1α; and vascularendothelial growth factor (VEGF) family, particularly VEGF (alsoreferred to in the art as VEGF-A), VEGF-B, VEGF-C, VEGF-D and placentalgrowth factor (PLGF).

Chemokines are a family of small proteins that play an important role incell trafficking and inflammation. Members of the chemokine familyinclude, but are not limited to, IL-8, stomal-derived factor-1(SDF-1),platelet factor 4, neutrophil activating protein-2 (NAP-2) and monocytechemo attractant protein-1 (MCP-1).

Other protein and chemical targets include: immunoregulation modulatingproteins, such as soluble human leukocyte antigen (HLA, class I and/orclass II, and non-classical class I HLA (E, F and G)); surface proteins,such as soluble T or B cell surface proteins; human serum albumin;arachidonic acid metabolites, such as prostaglandins, leukotrienes,thromboxane and prostacyclin; IgE, auto or alloantibodies forautoimmunity or allo- or xenoimmunity, Ig Fc receptors or Fc receptorbinding factors; G-protein coupled receptors; cell-surfacecarbohydrates; angiogenesis factors; adhesion molecules; ions, such ascalcium, potassium, magnesium, aluminum, and iron; fibril proteins, suchas prions and tubulin; enzymes, such as proteases, aminopeptidases,kinases, phosphatases, DNAses, RNAases, lipases, esterases,dehydrogenases, oxidases, hydrolases, sulphatases, cyclases,transferases, transaminases, carboxylases, decarboxylases, superoxidedismutase, and their natural substrates or analogs; hormones and theircorresponding receptors, such as follicle stimulating hormone (FSH),leutinizing hormone (LH), thyroxine (T4 and T3), apolipoproteins, lowdensity lipoprotein (LDL), very low density lipoprotein (VLDL),cortisol, aldosterone, estriol, estradiol, progesterone, testosterone,dehydroepiandrosterone (DHBA) and its sulfate (DHEA-S); peptidehormones, such as renin, insulin calcitonin, parathyroid hormone (PTH),human growth hormone (hGH), vasopressin and antidiuretic hormone (AD),prolactin, adrenocorticotropic hormone (ACTH), LHRH,thyrotropin-releasing hormone (THRH), vasoactive intestinal peptide(VIP), bradykinin and corresponding prohormones; catechcolamines such asadrenaline and metabolites; cofactors including atrionatriutic factor(AdF), vitamins A, B, C, D, E and K, and serotonin; coagulation factors,such as prothrombin, thrombin, fibrin, fibrinogen, Factor VIII, FactorIX, Factor XI, and vonWillebrand factor; plasminogen factors, such asplasmin, complement activation factors, LDL and ligands thereof, anduric acid; compounds regulating coagulation, such as hirudin, hirulog,hementin, hepurin, and tissue plasminigen activator (TPA); nucleic acidsfor gene therapy; compounds which are enzyme antagonists; and compoundsbinding ligands, such as inflammation factors.

Non-human derived targets and anti-targets include without limitation;drugs, especially drugs subject to abuse, such as cannabis, heroin andother opiates, phencyclidine (PCP), barbiturates, cocaine and itsderivatives, and benzodiazepine; toxins, such as heavy metals likemercury and lead, arsenic, and radioactive compounds; chemotherapeuticagents, such as paracetamol, digoxin, and free radicals; bacterialtoxins, such as lipopolysaccharides (LPS) and other gram negativetoxins, Staphylococcus toxins, Toxin A, Tetanus toxins, Diphtheria toxinand Pertussis toxins; plant and marine toxins; snake and other venoms,virulence factors, such as aerobactins, or pathogenic microbes;infectious viruses, such as hepatitis, cytomegalovirus (CMV), herpessimplex virus (HSV types 1, 2 and 6), Epstein-Barr virus (EBV),varicella zoster virus (VZV), human immunodeficiency virus (HIV-1, -2)and other retroviruses, adenovirus, rotavirus, influenzae, rhinovirus,parvovirus, rubella, measles, polio, pararyxovirus, papovavirus,poxvirus and picornavirus, prions, plasmodia tissue factor, protozoans,such as Entamoeba histolitica, Filaria, Giardia, Kalaazar, andtoxoplasma; bacteria, gram-negative bacteria responsible for sepsis andnosocomial infections such as E. coli, Acynetobacter, Pseudomonas,Proteus and Klebsiella, also gram-positive bacteria such asStaphylococcus, Streptococcus, Meningococcus and Llycobacteria,Chlamydiae Legionnella and Anaerobes; fungi such as Candida,Pneumocystis, Aspergillus, and Mycoplasma.

In one aspect the target includes an enzyme such as proteases,aminopeptidases, kinases, phosphatases, DNAses, RNAases, lipases,esterases, dehydrogenases, oxidases, hydrolases, sulphatases,cellulases, cyclases, transferases, transaminases, carboxylases,decarboxylases, superoxide dismutase, and their natural substrates oranalogs. Particularly preferred enzymes include hydrolases, particularlyalpha/beta hydrolases; serine proteases, such as subtilisins, andchymotrypsin serine proteases; cellulases; and lipases.

In another aspect the target is a stain on a fabric or other surfacematerial such as ceramic, glass, silica, wood, paper, metal and alloys,and living tissue, such as skin. The stain may be selected from thefollowing non-limiting group of stains; porphyrin derived stains, tanninderived stains, carotenoid pigment derived stains, anthocyanin pigmentderived stains, soil-based stains, oil-based stains, and human bodyderived stains. Particularly the stain may be a blood-derived stain or achlorophyll-derived stain. More specifically the stain may be grass;paprika; a tea-derived stain; or a fruit or vegetable derived stain,such as from wine, tomato and berries. A particularly preferred stain ishuman body soil, and more specifically stains referred to as collarsoil.

In yet another aspect the target includes hematopoietic stem cells(HSCs). A particularly preferred surface antigen expression profile ofHSCs is CD34⁺Thy-1⁺, and preferably CD34⁺Thy-1⁺Lin⁻. Lin⁻ refers to acell population selected on the basis of the lack of expression of atleast one lineage specific marker. Methods for isolating and selectingHSCs are well known in the art and reference is made to U.S. Pat. Nos.5,061,620; 5,677,136; and 5,750,397.

In a further aspect, preferred targets include cytokines, particularlyIL-2, IL-3, IL-6, IL-10, IL-12, IL-13, IL-14 and IL-16; EPO; GM-CSF; theTNF family; the VEGF family, GFIβ; and IL-1α. Cytokines are commerciallyavailable from several vendors including Amgen (Thousand Oaks, Calif.),Immunex (Seattle, Wash.) and Genentech (South San Francisco, Calif.).Particularly preferred are VEGF and TNF-α. Antibodies against TNF-α showthat blocking interaction of the TNF-α with its receptor is useful inmodulating over-expression of TNF-α in several disease states such asseptic shock, rheumatoid arthritis, or other inflammatory processes.VEGF is an angiogenic inducer, a mediator of vascular permeability, andan endothelial cell specific mitogen. VEGF has also been implicated intumors. Targeting members of the VEGF family and their receptors mayhave significant therapeutic applications, for example blocking VEGF mayhave therapeutic value in ovarian hyper stimulation syndrome (OHSS).Reference is made to N. Ferrara et al., (1999) Nat. Med. 5:1359 andGerber et al., (1999) Nat. Med. 5:623. Other preferred targets includecell-surface receptors, such as T-cell receptors.

It is preferred that the target and anti-target are characterized insome detail at the structural, chemical or genetic level to allow somecontrol over the purity, stability and concentration of the target.However, targets and anti-targets may be used that are not wellcharacterized. Non-limiting examples of potentially notwell-characterized targets include collar soil, tumor cells, human skinand hair.

A preferred anti-target includes fabric selected from the groupconsisting of cotton, wool, silk, polyester, rayon, linen, nylon andblends thereof.

In another aspect, when the target is damaged cells, tissue, or organs,the anti-target is healthy normal (non-damaged) cells, tissue, organs orcombinations thereof. Specific non-limiting anti-target examples includehealthy normal whole blood, skin, hair, teeth, and nails.

In some applications, the target and anti-target can be reverseddepending upon the specific application of interest. For example theremay be multiple applications where it is desirable to target human skinand not hair. Therefore the anti-target would be hair. In a similarapplication it may be desirable to target human hair and not thecorresponding anti-target, skin.

The following general examples of target/anti-target used in the sameapplication are provided for illustrative purpose only and are not meantto limit the selective targeting method disclosed herein: tumorcell/normal cell; receptor cell/cell not expressing the receptor;neoplastic cell/normal cell; soil stain/cotton fabric; foodstain/ceramic; specific protease/other protease; serine protease/wholeblood; hematopoietic stem cell/whole blood; specific enzymevariant/other forms of the enzyme; virus in a cell/cell; TNF-alpha/bloodcomponents; specific insect enzyme/homologous enzymes in animals;hematopoietic stem cell/other hematopoietic cells; hair/skin;nucleus/mitochondria; cytoplasm/nucleus; alpha/beta hydrolases/otherhydrolases; and a specific enzyme involved in photosynthesis/leaftissue.

Both the target and anti-target concentrations to be used in theselective targeting method will vary depending on the type of ligandlibrary, anti-target and target used. As discussed herein, the disclosedmethod has wide applicability to many different targets andanti-targets, therefore the concentration useful in the method may varyfrom about 1.0 M to 10⁻¹⁵M, preferably the concentration is in the 10⁻⁹M range. In general an excess amount of anti-target relative to theamount of target is required. While not meant to limit the invention,this excess amount may be in the range of at least 10 fold greater tomore than 1000 fold greater. An initial target concentration may bepreferably provided in the range of 10⁻³ M to 10⁻¹⁵ M. In one preferredembodiment, when the target is an enzyme, the target concentration maybe provided in the range of about 10⁻³ M to 10⁻¹² M. In anotherpreferred embodiment, when the target is a cytokine, the target may beprovided in the concentration range of about 10⁻³ M to 10⁻¹² M. In yetanother embodiment, when the target is a hematopoietic cell, the targetconcentration may be provided in the range of about 10 to 10⁹ cells.

In one preferred embodiment, when the anti-target is a blood protein oran enzyme, the anti-target concentration may be provided in aconcentration range of about 1.0 M to 10⁻¹² M.

In certain preferred embodiments, the anti-target or target may be amaterial or surface, such as a fabric, ceramic or micro-fluidic chip. Inthis instance the area of the target or anti-target will be important.While not intended to limit the invention in any manner, in general thesize of the anti-target or target material will be about 1.0 mm to 1.5cm; more preferably about 25.0 mm to 0.5 cm; however, the diameter orarea may be more or less than these values.

In one aspect, the invention is directed to the screening andidentification of ligands that bind to a selected target to form anon-covalent target-ligand complex with a binding affinity in the rangeof antibody affinities for antigens. The ligand binding affinityaccording to the present invention for K_(D), EC₅₀ or IC₅₀ is in therange of between about 10⁻⁷M to 10⁻¹⁵M, although higher or low bindingaffinities may be achieved. In one aspect, the affinity is in the rangeof at least about 10⁻⁷M, also at least about 10⁻⁵ M, preferably at leastabout 10⁻⁹M and also preferably at least about 10⁻¹²M. In anotherembodiment, the affinity is less than about 10⁻⁷M. In another aspect,k_(off) values for the ligand-target complex will be less than about10⁻³ sec⁻¹, less than about 10⁻⁴ sec⁻¹, and also less than about 10⁻⁵sec⁻¹. The ligands identified according to the selective targetingmethod of the invention will not bind with any significance to theanti-target. While not meant to limit the invention, a preferred ligandidentified according to the selective targeting method described hereinmay have a K_(D) for the anti-target greater than about 10⁻⁴ M, andpreferably greater than about 10⁻¹ M.

The selective targeting method according to the invention may becharacterized not only by the binding affinity of a ligand to thetarget, but also may be characterized by the selectivity of theligand-target complex. The selectivity of ligand binding for a targetcompared to ligand binding to an anti-target can be defined by a ratioof K_(D), EC₅₀ or IC₅₀ in the range of about 3:1 to 500:1. In oneaspect, selectivity is at least about 5:1, preferably at least about10:1, more preferably at least about 20:1, even more preferably at leastabout 30:1, even more preferably at least about 50:1, and yet morepreferably at least about 100:1.

In another aspect, the selective targeting method may be used to selectligands with a low affinity for the target but with a high selectivityfor the target. In this aspect, the selectivity of ligand bindingaffinity for the target compared to said ligand binding to ananti-target would be at least about 5:1, preferably at least about 10:1,also preferably at least about 20:1, more preferably at least about50:1, and even preferably at least about 100:1. However, the targetbinding affinity would be in the range of about 10⁻³M to 10⁻⁷ M.

Methods for measuring binding affinities and selectivity are well knownin the art, and these methods include but are not limited to measurementby radio-labeled release and competition assay; by isothermal titrationcalorimetry; biosensor binding assays (Morton & Myszka, (1998) MethodsEnzymol. 295:268-294); by fluorescence and chemi-luminescencespectroscopy; and by mass spectrophotometry (Gao et al., (1996), J. Med,Chem., 39:1949).

In one aspect, the anti-target is combined with the library of ligandsand allowed to incubate prior to exposing the library of ligands to thetarget. In another aspect, the anti-target and target are combined withthe library of ligands essentially simultaneously. Essentiallysimultaneously means at the same time or very close in time wherein theligand library is exposed to both the anti-target and the target priorto any separation step.

The selective targeting method as described herein may be performed invitro or in vivo. When performed in vitro, the library of ligands andthe anti-target (and optionally the target), are combined in or on avessel. The vessel may be any suitable material or receptacle such as aplate, culture tube, microtiter plate, micro-fluidic chip, petri dishand the like.

Preferably, the anti-target and the target are available in anenvironment where non-specific binding events are minimized. This may beaccomplished by various means including, but not limited to, 1) bycoating a vessel containing the ligand library and thetarget/anti-target with BSA, skim-milk or other adsorbing protein toblock non-specific binding, 2) by labeling the target molecule with acapture agent such as a biotinylated compound, for example biotin,avidin, or mutated form thereof which can be subsequently trapped bystreptavidin or a streptavidin derivative, such as nitrostreptavidin, 3)displaying the target/anti-target on magnetic beads that can bephysically separated from the library, or 4) by using library displayvectors with low background adsorption properties. These methods areknown in the art and reference is made to Parmley et al. (1988) supra;and Bayer et al., (1990) Methods Enzymol. 184:138.

A composition including a library of ligands and an anti-target may becombined together with additional compounds such as buffers andoptionally detergents and organic solvents under suitable conditions toallow binding of the ligands with the anti-target. One skilled in theart is well aware of useful buffers. Non-limiting examples include;tris(hydroxymethyl)aminomethane (Tris) buffers;N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffers;morphololino-ethanesulfonic acid (MES) buffers; buffered salinesolutions, such as N,N-bis[2-hydroxyethyl]2-aminoethanesulfonic acid(BES), Tris, and phosphate-buffered saline (PBS), preferably bufferedsaline solutions (Sambrook et al., (1989) supra). Commercial buffers areavailable for example SuperBlock™ (Pierce, Rockford, Ill.). Otheringredients such as detergents, for example Tween and Triton can be usedin the solutions.

Depending on the target, the composition including the ligand libraryand anti-target is incubated for a period of about 1 minute to about 96hours to allow the ligands to bind with the anti-target. However, longertime periods may be used depending on the stability of the target oranti-target. The component containing the unbound anti-target ligands isseparated from the anti-target bound ligands after incubation. While notessential, the separated component including the unbound anti-targetligands may optionally be transferred to a new vessel including theanti-target, incubated and then the component containing the unboundanti-target ligands can again be separated from the bound anti-targetligands. This transfer process may be repeated numerous times, forexample it may be repeated between 2 to 10 times or more. The repeatedtransfer step further reduces the number of ligands that bind to theanti-target. However, the contacting of the library of ligands with theanti-target and the separating of the anti-target bound ligands from theunbound ligands may be accomplished in one round. The contactingincluding incubation, and the separation steps, whether completed in oneround or in multiple rounds may generally be referred to as deselection.

In general, the temperature conditions during deselection may be between2 and 30° C. The temperature is limited by the stability of thecomponents and is well within the skill of one of ordinary skill in theart to determine.

The unbound anti-target ligands may be separated from the anti-targetbound ligands by methods well known in the art. Some of these methodsinclude liquid transfer, washing, centrifugation, filtration,chromatography, micro-dissection and fluorescence activator cell sorting(FACS).

The ligand library, depleted of anti-target binding ligands andcontaining unbound ligands is transferred to a vessel including thetarget under suitable conditions which will allow one or more members ofthe ligand library to bind with the target thereby forming atarget-bound ligand complex. In one aspect the ligands may be contactedwith the same target. In another aspect the ligands may be contactedwith an array of targets at the same time. One non-limiting example ofan array of targets includes the contacting of a ligand with multiplestains on a surface. The ligands are incubated under conditions thatallow binding to the target and generally for a period of time rangingfrom about 1 minute to about 96 hours. The incubation time depends onthe stability of the target. When the target is a stain, the incubationperiod will generally range from about 5 minutes to about 90 minutes.The vessel may further include buffers as described herein above. Thetemperature range is generally between about 2 and 30° C., andpreferably about 18 to 25° C.

One skilled in the art is well aware of references describing cell,organ, and tissue culture, and reference is made to Atlas and Parks(eds) (1993), The Handbook of Microbiological Media, CRC Press, BocaRaton Fla.; Gamborg and Phillips (eds) (1995) Plant Cell Tissue andOrgan Culture, Fundamental Methods, Springer Lab Manual Springer-Verlag.

The target-bound ligand complex may be subject to one or more washsteps. The washing compounds may include buffers (such as TBS and PBS),detergents, acids (glycine), organic solvents, bases, enzymes,sonication, or combinations thereof, wherein unbound ligands are washed.When the target-bound ligand complex is subject to an acid elution, thepH of the acid elution may be in the range of about 1.5 to 4.5,preferably in the range of about 2.0 to 3.5. The acid elution may takeplace for between 2 to 20 minutes and generally no longer than about 10minutes. The wash step may be repeated numerous times and in general canbe repeated between 2-6 depending on the specific target and ligandlibrary. Particularly when the washing step is with an acid, washingwill generally be followed by neutralization with various well-knowncompounds and buffers, such as TRIS-HCL. The washing step results in atarget-bound ligand complex comprising tight binding ligands having aK_(D), k_(off) and selectivity values as herein defined.

When the ligand library is contacted with the anti-target and targetessentially simultaneously as opposed to sequentially the ligandlibrary, anti-target and target composition may further include allmaterials described above for the sequential exposure of the anti-targetand target.

Further when the ligand library is contacted with the anti-target andtarget essentially simultaneously, the method may also be performed invivo. In this aspect, the library of ligands may be administered bymeans well known in the art, but preferably by injection into a host. Ifthe library is a phage-peptide library, the number of transducing unitsmay be in the range of 10⁴-10¹⁰. The host may be any animal, such as ahuman, mouse, chicken, or pig, preferably mouse. The target for examplemay be whole organs or damaged or tumor tissue, more specifically tumorblood vessels. If the target is a tissue or cells found in the blood,the library of ligands may be circulated in the blood for a period ofabout 1 minute to 10 minutes and allowed to bind with the target. Thetarget-bound ligand complex may be recovered after perfusion and thetissue dissected (Koivunen et al., (1999) Nature Biotech. 17:768 andArap et al., (1998) Science 279:377).

Separation of the target-bound ligands from the anti-target unboundligands or free ligands in the mixture may also be accomplished bywell-known means in the art and these methods include affinitychromatography; centrifugation; high-performance liquid chromatography(HPLC); filtration, such as gel filtration; enzyme-linked immunosorbentassays (ELISA); and fluorescence-activator cell sorting (FACS). Thechoice of the separating method will depend on various factors such asthe target, anti-target and ligand molecules. The choice of theseparation method is well within the skill of one in the art and avariety of instruments used for these separation methods arecommercially available. (See Kenny and Fowell (eds) (1992) PracticalProtein Chromatography Methods in Molecular Biology, vol. 11, HumanaPress, Totowa N.J.).

The target-bound ligand on the target-bound ligand complex may beidentified by various techniques including polymerase chain reaction(PCR), mass spectrophotometry (MS), surface plasmon resonance,immunoprecipitation and nuclear magnetic resonance (NMR) spectroscopy(U.S. Pat. No. 4,683,202; Szabo et al., (1995) Curr. Opin. Struct. Bio.)5:699; Harlow et al., (1999) Using Antibodies, A Laboratory Manual, ColdSpring Harbor Press; and Hajduk et al., (1999) J. Med Chem., 42:2315).Asymmetric PCR may also be used for identification of the target-boundligand wherein a single primer species or primers in differentialconcentration may be used. As well known to those in the art, when thelibrary members are genetically linked to the peptide or protein, DNA ormRNA can be amplified by PCR and the corresponding sequence subclonedinto a vector for sequencing and identification.

During the process of the identifying step, the target-bound ligand mayseparate from the target-bound ligand complex, but the identifying stepdoes not require separation, and preferably the target-bound ligand isnot separated from the target-bound ligand complex prior toidentification of the ligand. For example, in mass spectrophotometry(MS), once the target-bound ligand complex is injected into the massspectrophotometer the target-bound ligand may be separated from thetarget complex. Additionally, PCR may be directly carried out on thetarget-bound ligand complex.

The selective targeting method according to the invention preferablyincludes PCR to identify target-bound peptides. According to theinvention use of PCR results in the recovery of peptides not recoveredby conventional biopanning methods which utilize acid-elution. Ingeneral, a ligand encoding a DNA is amplified by PCR with appropriateprimers.

The presence of specific PCR products indicates that the target-boundligand encoding DNA is present. The amount of the target-bound ligand isdetermined by quantitative PCR. The degree of wash stringency can bemonitored to a desired level and to very low detection levels forexample to attomole levels. Nonspecific ligand binders may be competedout for example by adding wild type phage and designing primers thatonly amplify the ligand library. To prevent deterioration ofsignal-to-noise ratio, the sequences flanking the ligand encoding DNAmay be changed frequently during rounds of selection. Sensitivity forthe analysis of target-bound ligands may be controlled by changingtarget concentration, the number of PCR amplification cycles, thespecificity of the PCR primers, and the detection method for PCRproducts.

In one embodiment, when the target is a tumor antigen, tumor tissueincluding the target-bound phage ligands may be excised from a tumor andaddition of appropriate PCR primers, nucleotides, and polymerase mayyield the amplified PCR product. Various inhibitory reactions of PCR maybe alleviated by the addition of excipients including bovine serumalbumin, cationic amines, and organic solvents and reference is made toRoux, (1995) “Optimization and Troubleshooting in PCR” in PCR Primer: ALaboratory Manual, Cold Spring Harbor Press. DMSO and glycerol may beused to improve amplification efficiency and specificity of PCR. The DNAof the target-bound ligand may also be extracted and purified usingstandard techniques.

To facilitate sequencing of desired clones or separation from undesirednon-specific phage, the polynucleotide products generated by PCR may belabeled for example with biotinyl or fluorescent label moieties byincorporation during polymerase mediated catalysis. When the desired PCRproduct is to be cloned into a vector for additional rounds of selectivetargeting according to the method of the invention, it may be desirableto introduce diversity by mutagenic PCR methods, (See Stemmer, in Kay etal., supra). These include cassette mutagenesis, error prone PCR, DNAshuffling, ITCHY-SCRATCHY and the like as is well known by those in theart. Also reference is made to Tillett and Neilan, (1999) “Enzyme-freeCloning: A Rapid Method to Clone PCR Products Independent of VectorRestriction Enzyme Sites”: Nucl. Acids. Res., 27:26e.

As mentioned above and as well known in the art, the PCR fragments maybe cloned into various vectors for sequencing, they may be used in theformation of peptide protein fusions, or cloned into additional displayvectors.

The target bound library members may also be identified preferably bymass spectrometric methods. This is a rapid and accurate identificationof the structure of a compound based on the mass of the compound and onfragments of the compound generated in the mass spectrometry. The use ofmass spectrometry to identify the structure of compounds has beenreported in Cao et al., (1997) Techniques in Protein Chemistry VIII,Academic Press pages 177-184; and Youngquist et al., (1995) J. Am. Chem.Soc. 117:3900. Also reference is made to Cheng et al., (1995) J. Am.Chem. Soc., 117:8859 and Walk et al., (1999) Angew. Che. Int. Ed.,38:1763. One mass spectrometric technique is tandem mass spectrometry(MS/MS) wherein mass spectrometry is performed in tandem with liquidchromatography. To purify and separate the ligand of interest, this typeof MS is preferably used to screen target-bound ligands other thanphage-type peptides because of the need to separate and purifytarget-bound ligands from a biological system prior to injection of theligands into a mass spectrometer. Various recently developed MStechniques are available for identification of the target-bound ligands.(See Wu et al., (1997) in Chemistry and Biology, vol. 14(9):653,Marshall et al., (1998), Mass Spectrometry Reviews 17:1, and Nelson etal., (1999) J. Mol. Recognition, 12:77).

Following the screening of one or more ligand members, particularlypeptide ligands, the amino acid sequence of the peptides may bedetermined according to standard techniques known by those in the artsuch as direct amino acid sequencing of the selected peptide by usingpeptide sequencers, MS/MS, or manually or by determining the nucleotidesequence that encodes the peptide. The invention further includes thetarget-bound ligands, particularly the target-bound peptides identifiedaccording to the selective targeting method. Preferred target-boundpeptides identified according to the method include peptides having theamino acid sequence of SEQ ID NOs: 3-17; SEQ ID NOs: 18-26; SEQ ID NOs:29-49; SEQ ID NOs: 50-63; SEQ ID NOs: 64-77 and SEQ ID NOs: 79-102.

When multiple ligands are selected from the initial ligand library, andthe library is a peptide library, the amino acid sequences of theligands when aligned do not necessarily exhibit a conserved region or apeptide motif, which is herein defined as an amino acid consensussequence that represents preferred amino acid sequences in all of theselected peptides.

In a particular embodiment, the method concerns selecting peptides froma peptide library having a binding affinity for a target of betweenabout 10⁻⁷M to about 10⁻¹⁰ M which comprises, contacting a peptidelibrary with an anti-target to allow the peptides in the library to bindwith the anti-target; separating unbound peptides from the anti-targetbound peptides; contacting the separated unbound peptides with a targetunder conditions allowing binding of the unbound peptides with thetarget to form a target-bound peptide complex; separating thetarget-bound peptide complex from the peptides that do not bind to thetarget; and identifying the bound peptides on the target-bound peptidecomplex wherein the peptides are less than about 50 amino acids inlength, are not antibodies, and have a selectivity in the range of about10:1 to about 50:1. Preferably the peptides identified on thetarget-bound peptide complex are less than 25 amino acids in length withselectivity in the range of about 20:1.

Once the target-bound ligands are identified, the ligands may be exposedto repeated rounds of the selective targeting method and reference ismade to FIG. 1. The target-bound ligands may be subject todiversification. Diversification including chemical diversity mayinclude a number of mutagenesis techniques. See Saiki et al., (1988)Science 239:487; Zoller et al., (1982) Nucl. Acids. Res. 10:6487; andSmith (1985) Ann Rev. Genetics. 19:423. The target-bound ligands may besequenced to determine the identity of the bound ligands and thenoligonucleotides may be made based on the sequences but which includesmall variations. PCR may be used to make small changes in thenucleotide coding sequences for the ligands. This PCR mutagenesis canresult in a mutation at any position in the coding sequence.Diversification may also take place by mutagenesis of a small subset ofidentified ligands. In general diversified ligands will have at least80%, 85%, 90%, 95%, 97% or 99% sequence identity at the nucleotide levelto the target-bound ligand. When the ligand is a peptide the diversifiedpeptide will have at least 80%, 85%, 90%, 95%, 97% or 99% amino acidsequence identity to the identified target-bound peptide. Thediversified ligands may be exposed to one or more rounds of theselective targeting method of the present invention. The diversifiedligands may be screened with other identified target-bound ligands fromwhich they were derived and assayed in appropriate applications forwhich the ligands were originally screened.

The selective targeting method of the current invention for screening alibrary of ligands that bind to a target has wide utility for manyapplications. In one particular application, the selective targetingmethod described herein may be used to identify ligands that bind to atarget under harsh conditions. A harsh condition may include but is notlimited to acidic pH, high temperature, and exposure to detergents, suchas those found in household laundry detergents. In this respect, oneexemplary application according to the invention is screening andidentification of a ligand, particularly a peptide, which is useful incleaning applications. Cleaning applications include but are not limitedto detergent compositions, stain removal compositions, and textiletreatment compositions. Particular stain targets include human body soilstain, a porphyrin derived stain, a tannin derived stain, a carotenoidpigment derived stain, an anthocyanin pigment derived stain, asoil-based stain, or an oil-based stain. Components of various cleaningcompositions and particularly detergent compositions, are well known inthe art and are not repeated herein in any detail. The compositions mayinclude, but are not limited to one or more of the following components;surfactants, such as; anionic, nonionic, cationic, amphoteric, soaps andmixtures thereof; builders, such as; phosphate builders, for exampletriphosphates, sodium aluminosilicate builders, for example zeolites;organic builders, for example polycarboxylate polymers; enzymes, such asproteases, cellulases, lipases and others; enzyme-stabilizers; bleachingagents; dyes; masking agents; softening agents; and others. Reference ismade to the following references U.S. Pat. Nos. 3,929,678; 4,760,025;4,800,197; 5,011,681; and McCutheon's Detergents and Emulsifiers, NorthAmerican Edition (1986) Allured Publishing Co.

In another particular application, selective targeting according to theinvention may be used to screen and identify a ligand useful fortherapeutic intervention. In this respect a library of ligands may bescreened to identify a tumor-bound ligand. The tumor may be a carcinoma,sarcoma or melanoma. While one skilled in the art could envisage anynumber of anti-targets one preferred anti-target is a normal cell. Oncea tumor-bound ligand is identified the ligand may be used to preventtumor cell migration, tumor cell establishment and/or tumor cell growthin vivo.

In yet another particular therapeutic intervention application a libraryof ligands may be screened according to the invention to identify acytokine and in particular a TNF or a VEGF. A cytokine-bound ligand mayprevent the cytokine from binding with its corresponding receptor. Thisinhibition could render the cytokine inactive and inhibit downstreamsignal transduction that controls various disease states. While oneskilled in the art could envisage any number of anti-targets, onepreferred anti-target is blood. Another preferred anti-target is thecorresponding receptor or an isoform.

In a further application, the selective targeting method according tothe invention may be used to identify ligands, particularly peptides,useful in personal care applications for example skin care or hair care.

In another application, the selective targeting method according to theinvention may be used to identify cell type specific surface molecules.Preferred anti-targets include one or more different cell types, cellsin different states, or cells that do not display the surface molecule.

The selective targeting method and the ligands identified according tothe method may be used in broad applications. In addition to theapplications discussed herein above, other non-limiting applications,particularly for peptide ligands include: 1) for mapping antibodyepitopes; 2) in providing new ligands for important binding molecules,such as enzymes and hormone receptors; 3) in providing potentialagricultural compounds with pesticidial properties; 4) for developingnew drug leads and exploiting current leads; 5) identifying industrialcatalysts; 6) in identifying highly sensitive in vivo and in vitrodiagnostic agents; 7) for increasing the efficiency of enzyme catalystsby binding metals and other cofactors; 8) for controlling proteaseaction in vivo; 9) to change inhibitory properties of targeted proteins;10) use in developing a targeted enzyme; 11) use in selective deliveryof gene therapy vectors to specific tissues or cell types; and 12) usein drug delivery or targeted actives.

Accordingly, the following examples are offered by way of illustration,and are not meant to limit the invention in any manner. Those skilled inthe art will recognize or be able to ascertain using no more thanroutine experimentation, many equivalents to the specific embodiments ofthe invention described herein.

EXAMPLES

The procedures for restriction digest, ligation, preparation ofcompetent cells using calcium chloride, preparation of 20 mg/mlisopropyl (IPTG), preparation of 20 mg/ml5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal), and preparation ofphosphate-buffered saline (PBS) were according to well-known methods inthe art and can be found in Sambrook et al. (1989) supra.Phage-displayed libraries (cyclic 7-mer, linear 7-mer and linear 12-mer)were supplied by New England Biolabs ((NEB; Beverly, Mass.). Restrictionendonucleases EagI and Acc65I, 10× NEBuffer 3, T4 DNA ligase, alkalinecalf intestinal phosphatase, E. coli ER2537 host strain, and M13KE gillcloning vector were supplied by NEB and used according to themanufacturer's instructions unless stated otherwise. Taq polymerase,10×PCR Buffer, and dNTP mix were supplied by Roche MolecularBiochemicals (Indianapolis, Ind.). PCR was carried out using a HYBAIDOmn-E Thermocycler from E&K Scientific Products (Campbell, Calif.).

Both the QIAquick Gel Extraction Kit and QIAquick PCR Purification Kitwere obtained from QIAGEN (Valencia, Calif.). AmpliWax™ PCR Gems wereobtained from Perkin Elmer. Phenol/chloroform extractions were carriedout using Phase Lock Gels™ I (light) from 5 Prime 3 Prime, Inc.(Boulder, Colo.). Nondenaturing Polyacrylamide Gels (8%) and D-15 DNAMarkers were obtained from Novex (San Diego, Calif.).

Example 1 Selection of Phage-Peptides that Bind to Tumor Necrosis Factorα (TNF-α) Using PCR for Identification of High Affinity Phage-PeptideClones

A thin-walled PCR tube was coated with the target human (h)TNF-α(BioSource International; Camarillo, Calif.) by incubating 100 μl of 0.5mg/ml purified TNF-α in PBS overnight at 4° C. in the PCR tube. Excessunbound TNF-α was removed, and the tube was coated overnight at 4° C.with 100 μl of SuperBlock™ blocking buffer (Pierce: Rockford, Ill.) inTris buffered saline (TBS). The anti-target (SuperBlock™ blockingbuffer) was prepared in separate PCR tubes coated overnight at 4° C.with 100 μl of SuperBlock™. A phage-displayed 7-mer random peptidelibrary (10 μl of 2×10¹³ plaque forming units (pfu)/ml) was diluted in50 μl of PBS and incubated at 4° C. with shaking for 30 minutes in theanti-target PCR tube. The supernatant was transferred to anotheranti-target PCR tube and this procedure was repeated 3 times to greatlyreduce the number of phage-displayed peptides that bind to theanti-target.

The supernatant containing the phage-peptide library (depleted ofanti-target binders) was transferred to the target PCR tube coated withTNF-α and incubated for 4 hours at 4° C. with shaking to allow thephage-displayed peptides to bind to the target. Unbound phage wereremoved by washing the tube 5 times with 150 μl PBS containing 0.1%Tween-20 at room temperature. Low affinity binders were washed away byincubating with 60 μl of 0.2 M Glycine (pH 2.2) for 6 minutes followedby neutralization with 9 μl 1M Tris-Cl (pH 9.1). The acid washedpopulation was retained for further analysis. The tube was then washedagain 3 times with 150 μl of PBS.

To the remaining phage-peptides bound to the TNF-α, 54 μl of LysisBuffer A (10 mM Tris-Cl, pH 8.4, 0.1% Triton-X100) and an AmpliWax™ PCRgem (Perkin Elmer, Norwalk, USA.) was added. The tube was heated at 95°C. for 15 min and then allowed to cool. The following PCR reagents werethen added:

10 mM dNTPs 2.5 μl 50 μM CMM13-01 primer 10 μl 50 μM CMM13-02 primer 10μl 10X PCR Buffer 7.5 μl Taq Polymerase (5 U/ml) 1 μl

PCR amplification was performed using 20 cycles of denaturation at 94°C. for 15 sec, annealing at 55° C. for 20 sec, and extension at 72° C.for 30 sec. The sequences of the primers (synthesized by GIBCO BRL)were:

SEQ ID NO: 1 CMM13-01 5′ CCTCGAAAGCAAGCTGATAAC 3′ SEQ ID NO: 2CMM13-02 5′ CATTCCACAGACAACCCTCATAG 3′

The PCR product (267 base pairs (bp)) was analyzed on an 8%polyacrylamide gel (FIG. 2, third lane) along with the PCR product froma single phage peptide clone (positive control, FIG. 2, second lane) andmolecular weight markers (FIG. 2, first lane). A slower running productthat appeared as a diffuse band was observed at around 500-700 bp. Thiswas due to too much template (i.e. phage) in the PCR reaction, and canbe alleviated by decreasing the phage concentration or by decreasing thenumber of PCR cycles (see FIG. 3 below). To decrease the amount of the500-700 bp diffuse band, the PCR product was diluted appropriately forsubsequent PCR reactions from this starting material to generate moreproducts for sub-cloning purposes. Once the desired product (267 bp) wasamplified, it was digested with EagI and Acc65I restrictionendonucleases to produce a 45 bp fragment containing the DNA coding forthe random peptide. The 45 bp fragment was then sub-cloned into theM13KE vector (New England Biolabs; Beverly, Mass.) at the EagI andAcc65I restriction sites using standard techniques (Sambrook, et al.,(1989) supra). After ligating the 45 bp fragment into the M13KE vector,the ligation reaction was transformed into chemically competent ER2537E. coli cells. The cells were made competent with calcium chloride usinga standard protocol (Sambrook, et al., (1989) supra). The M13 DNA wasisolated from various transformants using a modified protocol from NewEngland Biolab's protocol for M13 DNA preparation and then sequenced.The modification includes the use of 96-well plates as opposed to tubes.The corresponding peptide sequences are shown in Table 1.

TABLE 1  Amino Acid sequences that bind to TNF-alphaand not to SuperBlock ™ Clone Amino Acid ID Sequence Frequency^(a) T1 RYWQDIP 8 SEQ ID NO: 3 T2  APEPILA 7 SEQ ID NO: 4 T3  DMIMVSI 3SEQ ID NO: 5 T4  WTPKPTQ 2 SEQ ID NO: 6 T5  ATFPNQS 2 SEQ ID NO: 7 T6 ASTVGGL 2 SEQ ID NO: 8 T7  TMLPYRP 2 SEQ ID NO: 9 T8  AWHSPSVSEQ ID NO: 10 T9  LTQSFSS SEQ ID NO: 11 T10 THKNTLR SEQ ID NO: 12 T11GQTHFHV SEQ ID NO: 13 T12 LPILTQT SEQ ID NO: 14 T13 SILPVSHSEQ ID NO: 15 T14 LSQPIPI SEQ ID NO: 16 T15 QPLRKLP SEQ ID NO: 17^(a)Number of multiple times this amino acid sequence occurred out of 24clones sequenced

Example 2 Characterization of Binding Affinity & Selectivity ofPhage-Peptides that Bind to TNF-α

The binding and dissociation of phage clone A1, amino acid sequence:RYWQDIP; Table 1, (SEQ ID NO: 3) to TNF-α was monitored using an IAsysAutoPlus Biosensor following the Labsystems Affinity Sensors IAsysProtocol 2.4 ‘Immobilization to Protein Layer: Thiol coupling to avidin’(Thermo BioAnalysis Corp. Franklin, Mass.). Two cuvettes were firstcoated with avidin and one (the control) was then blocked with biotin.This was followed by activation of the lysine groups. A 15 μl aliquot ofa 1 mg/ml (h)TNF-α solution was added to each cuvette. No binding of theprotein to the surface was observed in the control cuvette, but (h)TNF-αwas clearly immobilized on the unblocked avidin-coated cuvette (notshown). This complex was stable and did not dissociate over a 10 minutetime period. After blocking and washing, phage clone A1, RYWQDIP (SEQ IDNO: 3) was added to give a final titer of 5×10¹¹ pfu/ml. As shown inFIG. 4A, there is significant binding of the phage to the TNF-α in thesample cuvette, while very little phage bound to the control cuvette.

The dissociation of the phage from the TNF-α is very slow with thedissociation constant estimated as k_(off)<10⁻⁴ sec⁻¹ (10 mM HEPES/0.05%Tween). Washing with a 10× buffer concentrate (at the 70 min time point)only removed a small portion of the phage. Additional washes with 10 mMHCl failed to completely remove the phage peptide from the target.Binding of phage-displayed peptide sequence RYWQDIP (SEQ ID NO: 3) isspecific since wild-type phage lacking the insert did not bind toimmobilized TNF.

Example 3 Selection of Phage-Peptides that Bind to IL-6 and IL-8

Using the same method as described in Example 1, human IL-6 and IL-8were used as targets and SuperBlock™ blocking buffer was used as ananti-target. The PCR tubes were coated with recombinant human IL-6 (0.1mg/ml) and IL-8 (0.25 mg/ml) (Biosource International). Selectionsyielded PCR bands of the expected size (267 bp) even after acid elutionof phage from the target (FIG. 2, fourth and fifth lanes, respectively).

Example 4 Selection of Phage-Peptides that Bind to VEGF

A sterile microtitre plate (5 wells/sample) was coated with 200 μl 1%PBS/BSA (PBS+1% Bovine Serum Albumin) followed by washing 3× with 200 μl0.25% PBST. The wells were left filled. A library of phage peptides weredeselected against whole human blood as the anti-target by mixing 100 μlfresh, whole human blood with 10 μl phage library, adding to the firstcoated well, and incubating for 30 minutes at room temperature (RT).Following 30 minutes, the solution was aspirated and delivered to thenext coated well. This procedure was repeated 4 times to generate thelibrary of anti-target non-binding phage. For the target, 5 mg of 200 μmpolystyrene beads were coated with human VEGF, by incubating with 100 μlof 100 μg/ml recombinant human VEGF (Biosource International; Camarillo,Calif.) overnight at 4° C. with gentle agitation. Excess unbound VEGFwas removed by washing 3 times with PBST (0.25% Tween-20 in 1×PBS). Thebeads were then blocked with 2% Tween-20 a×PBST for two hours at roomtemperature (RT). A phage-displayed cyclic 7-mer random peptide librarywas used. The selection procedure is essentially the same as describedin Example 1. After the first round of selection, the PCR fragment fromthe target-bound ligand was purified, digested with EagI and Acc651, andthe restriction enzymes were heat denatured. The fragments were ligateddirectly into M13KE cut vector using the Takara ligation Kit (PromegaCorp). Ligation mixes were transformed, and amplified according tostandard procedures (Sambrook et al. (1989) supra). A second round ofselection was carried out to further enrich phage-peptides that bind toVEGF. The corresponding peptide sequences are shown in the followingtable:

TABLE 2  Amino Acid sequences that bind to VEGF usingselective targeting Clone ID Amino Acid Sequence SEQ ID NO: V-1 CSKHSQITC SEQ ID NO: 79 V-2  CKTNPSGSC SEQ ID NO: 80 V-3  CRPTGHSLCSEQ ID NO: 81 V-4  CKHSAKAEC SEQ ID NO: 82 V-5  CKPSSASSC SEQ ID NO: 83V-6  CPVTKRVHC SEQ ID NO: 84 V-7  CTLHWWVTC SEQ ID NO: 85 V-8  CPYKASFYCSEQ ID NO: 86 V-9  CPLRTSHTC SEQ ID NO: 87 V-10 CEATPRDTC SEQ ID NO: 88V-11 CNPLHTLSC SEQ ID NO: 89 V-12 CKHERIWSC SEQ ID NO: 90 V-13 CATNPPPMCSEQ ID NO: 91 V-14 CSTTSPNMC SEQ ID NO: 92 V-15 CADRSFRYC SEQ ID NO: 93V-16 CPKADSKQC SEQ ID NO: 94 V-17 CPNQSHLHC SEQ ID NO: 95 V-18 CSGSETWMCSEQ ID NO: 96 V-19 CALSAPYSC SEQ ID NO: 97 V-20 CKMPTSKVC SEQ ID NO: 98V-21 CITPKRPYC SEQ ID NO: 99 V-22 CKWIVSETC SEQ ID NO: 100 V-23CPNANAPSC SEQ ID NO: 101 V-24 CNVQSLPLC SEQ ID NO: 102

To compare the selective targeting method of the current invention withthe conventional biopanning method, a parallel experiment usingconventional acid-elution method was performed. Three rounds ofbiopanning according to methods described by Smith and Scott (1990)Science 249:386 yielded the sequence profiles summarized in Table 3.These sequences do not overlap with the sequences identified theselective targeting method according to the current invention (Table 2).

TABLE 3  Amino Acid sequences that bind VEGF usingconventional biopanninq method Clone ID Amino Acid Sequence SEQ ID NO.BP 81 CYNLYGWTC SEQ ID NO: 103 BP 82 CTLWPTFWC SEQ ID NO: 104 BP 83CNLWPHFWC SEQ ID NO: 105 BP 84 CSLWPAFWC SEQ ID NO: 106 BP 85 CSLWPHFWCSEQ ID NO: 107 BP 86 CAPWNSHIC SEQ ID NO: 108 BP 87 CAPWNLHICSEQ ID NO: 109 BP 96 CLPSWHLRC SEQ ID NO: 110 BP 97 CPTILEWYCSEQ ID NO: 111 BP 02 CTLYPQFWC SEQ ID NO: 112 BP 04 CHLAPSAVCSEQ ID NO: 113

Example 5 Selection of Phage-Peptides that Bind to Collar Soil

Soiled shirt collars on cotton or 65% polyester:35% cotton containingthe target (collar soil) and anti-target EMPA 213 polyester cottonfabric (Test Fabrics, Freehold, N.J.)) were cut to a diameter of 7/32″using a die with an expulsion to fit an NAEF punch press (MS InstrumentCompany, Stony Creek, N.Y.). A 96-well flat bottom microtiter plate(Costar, cat #3598) was coated overnight with SuperBlock™ blockingbuffer and then washed with 200-250 μl TBST (0.1% Tween-20) using anEL403 auto plate washer (Bio-Tek Instruments, Winooski, Vt.). The fabricpieces were placed in the wells and a 10 μl stock solution of aphage-peptide 12-mer library displayed on M13 filamentous phage wasadded to 100 μl of detergent (3.4 g/L European Detergent) in a wellcontaining polyester-cotton as the anti-target. After a 20-minuteincubation, the supernatant containing unbound phage (anti-targetnon-binders) was transferred to a second well containing thepolyester-cotton fabric. This was repeated once more. The supernatantwas then transferred to the well containing the soiled shirt collarfabric, and the remaining phage peptide population was selected for“stain binders” by incubation with the stain for 10-60 min. The stainwas subjected to a series of wash steps with either TBST containing0.1-2% Tween-20 or 3.4 g/L detergent. The wash step can be manipulatedtoward the desired stringency. After an initial wash in the originalwell containing the stain, the stained fabric piece containing anyremaining bound phage was transferred to the next well for a second washstep.

A portion of the stained fabric containing the bound phage wastransferred to a PCR tube with 60 μl of Lysis Buffer B (10 mM Tris-Cl,pH 8.4, 1% Triton-X100; 10 mM EDTA) and an AmpliWax™ PCR gem (PerkinElmer, Norwalk, USA.). The tube was heated at 95° C. for 20 min and thenallowed to cool. PCR amplification of target-bound phage was carried outas described in Example 1 with minor modification. FIG. 3B showsamplification of a single band of homoduplex DNA requires less than 20PCR cycles (second lane in FIG. 3B). Longer cycle times (first lane inFIG. 3B) yield substantial fractions of heteroduplex DNA formationwhereas shorter cycle times (third lane in FIG. 3B) do not yieldmeasurable PCR products. The correct size PCR product was gel purifiedon a 8% polyacrylamide gel, subcloned back into M13KE and sequenced asdescribed in Example 1. The amino acid sequences corresponding to phagepeptide clones that bind to collar soils and not to polyester cottonfabric in detergent are summarized in Table 4.

TABLE 4  Amino Acid sequences that bind to collarsoils and not to polyester-cotton Clone Amino Acid ID SequenceFrequency^(a) C1  HPASQTFTFTRT 2 SEQ ID NO: 18 C2  NSDVLFKPYPMF 7SEQ ID NO: 19 C4  SISSTPRSYHWT 8 SEQ ID NO: 20 C8  TPSTMPPSLPLRSEQ ID NO: 21 C14 TPDKDTMSPPVP SEQ ID NO: 22 C16 HLPVRITDWFHHSEQ ID NO: 23 C19 EPILMRASPFRE SEQ ID NO: 24 C20 ESSAFTALSGQPSEQ ID NO: 25 C21 SSPNMITLLSSL SEQ ID NO: 26 ^(a)Number of multipletimes this amino acid sequence occurred out of 24 clones sequenced.

Example 6 Selective Binding of a Radiolabeled Peptide to Target CollarSoils on Fabric

Soiled shirt collars (cotton or 65% polyester:35% cotton) containing thetarget (collar soil) and anti-target EMPA 213 polyester cotton (TestFabrics, Freehold, N.J.)) were cut to a diameter of ½″ and placed into aCostar 24-well plate. The soil targeting peptide SISSTPRSYHWT (SEQ IDNO: 20) identified according to Example was N-terminally labeled with¹⁴C-glycine. 10 μL of a 400 μM solution of [1-¹⁴C-G]SISSTPRSYHWT(SynPep, Dublin, Calif.) (SEQ ID NO: 114) was added to 4 mL of 50 mMCAPS buffer, pH 10.4, containing 0.002% Tween-20. 950 uL aliquots of theradiolabeled peptide were added to each well and samples were shaken ona rotary shaker at 30° C. for 30 minutes. Samples were removed andwashed with 4 mL of buffer, followed by 4 mL of milliQ H₂O for 20 min.Samples were air dried on Whatman filter paper, and digitally scannedwith an Hewlett Packard scanner (Palo Alto, Calif.). The radioactivelylabeled swatches were then exposed to a phosphor screen (MolecularDynamics; Sunnyvale, Calif.) for 30 hours at −70° C. The resultingphosphorimage was scanned using a Molecular Dynamics Storm® system. FIG.5 illustrates the visual image of the target (stain) and anti-targetalong with the corresponding phosphorimage of stained and controlfabric. The relative intensity of the phosphorimage was quantitatedusing the ImageQuant® image analysis software (Molecular Dynamics;Sunnyvale, Calif.) and shows that the selectivity ratio of stain bindingto fabric binding is >15:1.

Example 7 Demonstration of Slow K_(off) Rate Constant for Release ofStain Targeted Peptide

Soiled shirt collars (65% polyester: 35% cotton) containing the target(collar soil) and anti-target along with the control anti-target(unstained polyester:cotton from the same shirt) were cut to a diameterof 7/32″ and placed into a 96-well microtiter plate (Millipore Corp.,0.22 μM Durapore membrane; Cat. No. MAGV N22 50). Serial dilutions of a400 μM stock solution of a soil targeting peptide SISSTPRSYHWT (SEQ IDNO: 20) and a peptide control NFFPTWILPEHT (SEQ ID NO: 78) bothterminally labelled with ¹⁴C-glycine were added to 1 g/L Tide detergentsolutions (Procter and Gamble, Cincinnati, Ohio) and 60 μL aliquots wereplaced into the wells of the microtiter plate. The plate was incubatedwith shaking for 30 minutes at 32° C. followed by suction filtering ofexcess unbound radiolabeled peptide (Vacuum manifold; Millipore Corp.Cat. No. MAVM 0960R). The samples were rinsed three times with 200 μL ofdistilled water, with shaking in between rinses, over a period of about40 minutes. The remaining radioactivity bound to the samples wasquantitated by liquid scintillation counting in a Wallac microbetacounter. FIG. 6A shows that greater than 50% of the total radiolabelremains bound to the stained fabric for the stain targeted peptide, evenafter rinsing for 40 minutes. This corresponds to a rate constant forrelease of the soil targeting peptide k_(off)≦2×10⁻⁴ sec⁻¹. In contrast,the control peptide shows no affinity or selectivity in the same assay,FIG. 6B.

Example 8 Selectivity and Affinity of Peptides for Acid Elution Comparedto the Selective Targeting Method of the Invention

A phage peptide sequence HTFQHQWTHQTR, (SEQ ID NO: 27) that binds tocollar soil on cotton was identified after five rounds of biopanning asdescribed in example 5 except that phage peptides were eluted by acidafter each round using the methods described in Scott and Smith (1990)Science 249:386. The selectivity (stain vs. cotton binding) and affinity(k_(off)) were measured for the corresponding peptide binding to collarsoil as follows: A 1 mM solution of Ni chelated GGHTFQHQWTHQTR (SEQ IDNO: 28) was incubated with collar soil on cotton or cotton alone for 90minutes at room temperature with shaking in a microtiter plate. Acontrol peptide chelate Ni GGH was also tested under the sameconditions. After pipeting off the incubation solution from the well,fabric swatches were rinsed in 200 μL water with shaking for 3 minutes.The residual bound peptide was assayed by adding 200 μLo-phenylenediamine (OPD) and 50 μL of 100 mM H₂O₂ and measuring theabsorbance of the oxidized OPD at 420 nm. As summarized in Table 5 andFIG. 7, the selectivity ratio of stain binding to fabric is less than orequal to ≦3:1 and the affinity as measured by k_(off)=1×10⁻³ sec⁻¹.These data demonstrate specific and selective tight binding peptides arepreferably identified using the selective targeting methods according tothe present invention.

TABLE 5  Summary of Collar Soil Binding Peptide Selectivity and AffinityMethod of Rounds of Selectivity Affinity Sequence identificationSelection Ratio 10⁻⁴ k_(off) (sec⁻¹) GSISSTPRSYHWT Selective 1 >15:1 ≦2SEQ ID NO: 114 Targeting (PCR) GGHTFQHQWTHQTR Biopanning 5  ≦3:1 10SEQ ID NO: 28

Example 9 Stability of Phage Displayed Libraries in a Detergent Matrix

To examine the effect of household laundry detergents on the stabilityof phage peptide libraries, a stock solution of a peptide 12-mer librarydisplayed on M13 filamentous phage (New England Biolabs, Beverly Mass.,USA) containing 10¹³ pfu/mL was diluted to 10¹² pfu/mL in a) 100 mM TrisHCl, pH 7.5, 0.1% Tween-20 (TBST control) b) 0.7 g/L of Ariel Futur(Procter & Gamble, Cincinnati, Ohio) containing 3 grams per gallon (gpg)hardness, and c) 3.4 g/L of Ariel Futur containing 15 gpg hardness.Aliquots (100 μL) were added to the wells of a 96-well flat bottommicrotiter plate (Costar, cat #3598) that was blocked with Superblockblocking buffer in TBS (Pierce, cat #37535). The samples were incubatedat 25° C. with gentle rocking for 90 minutes. 10 μL aliquots wereremoved and serially diluted into Luria Broth for phage titteringaccording to standard procedures (Kay et al., (1996) supra). No loss inphage titer was observed in detergent solution, relative to the controlphage library in TBST.

Example 10 Selection of Phage-Peptides that Bind to Polyurethane and notto Cotton, Polyester, or Polyester-Cotton Fabrics

1.5 mL microfuge tubes were blocked overnight with Blocking buffer-PBS,washed with 1 mL 3.4 g/L detergent and drained. 500 μL 3.4 g/L detergentwas added to 4 tubes, along with one piece each cotton, polycotton, andpolyester fabric. Phage peptide libraries were added as follows:

Tube 1 10 μL Ph. D.-C7C library Tube 2 10 μL Ph. D.-12 library Tube 3 10μL wild-type phage control Tube 4 no phage control

The tubes were incubated at room temperature for 20 minutes at 1000 rpmin an Eppendorf thermomixer and fabric pieces were removed. Thisdeselection step was repeated at total of 3 times, followed byincubation of the phage libraries with a polyurethane plug wetted bysqueezing with a clean pipet tip for 30 minutes.

The supernatant was aspirated from the plugs using a clean pipet tipattached to a vacuum line and 1 mL of 3.4 g.L detergent solution wasadded to the tube. The plug was rewetted by squeezing with cleaninoculation loop, and tubes were placed in the Eppendorf thermomixer fora total of 10 washes. Plugs were transferred to clean 100 mL disposablefilter systems(Corning) and 3×40 mL PBST (0.25% v/v Tween-20) washeswere performed by delivering the wash solution to the filter system.Plugs were rewetted by squeezing with a clean pipet tip, incubatedmomentarily with the PBST, then dried by aspiration. Ten 1 mL PBS washeswere performed by pipetting wash solution directly onto the plug whilethe filter system was under vacuum.

The plugs were transferred to clean 0.5 mL microfuge tubes. 100 μL lysisbuffer (0.1% Triton X-100, 10 mM Tris pH 8.4) was added, and the tubeswere incubated at 95° C. for 20 minutes to lyse the phage. Lysed phagewere PCRed in the same tube as follows:

-   -   50 μL HotStarTaq® master mix (QIAGEN)    -   25 μL lysis buffer    -   5 μL BSA (10 μg/μL)    -   1.25 μL CMM13-01 primer (50 μM)    -   1.25 μL CMM13-02 primer (50 μM)    -   17.5 μL H₂O

PCR amplification was performed using 30 cycles of denaturation at 95°C. for 15 sec, annealing at 58° C. for 30 sec, and extension at 72° C.for 30 sec followed by a single cycle at 72° C. for 5 min. PCR productswere cloned into the TOPO®-10 vector (Invitrogen, San Diego, Calif.) byzero blunt cloning according to the manufacturer's instructions. Cloneswere sequenced using standard sequencing methods and summarized in Table6.

TABLE 6  Amino Acid sequences that bind topolyurethane and not to fabrics Clone ID Amino Acid Sequence P 39HPSWAPVSSTLR SEQ ID NO: 29 P 40 STPHQPCATAPH SEQ ID NO: 30 P 41LDQILTSSRIWP SEQ ID NO: 31 P 42 HYLKNVEATGPR SEQ ID NO: 32 P 43SSRMYPSPDSFM SEQ ID NO: 33 P 44 SMATQLQGNITM SEQ ID NO: 34 P 45YMHASLMWAFG SEQ ID NO: 35 P 46 KALPPNSTLSRA SEQ ID NO: 36 P 47LELPNNIQSITS SEQ ID NO: 37 P 48 QVFHIAGVRDQV SEQ ID NO: 38 P 49REPAPSCTTTCL SEQ ID NO: 39 P 50 YPHHPRLHYTFS SEQ ID NO: 40 P 52KVTEFQKAHCSS SEQ ID NO: 41 P 53 GITLHNTMVPWT SEQ ID NO: 42 P 54EAGLSPTRPYMF SEQ ID NO: 43 P 56 SHHTHYGQPGPV SEQ ID NO: 44 P 57FYPSPSTAKMWR SEQ ID NO: 45 P 58 SGFQSAYAFPYS SEQ ID NO: 46 P 59MVSQPDPRATLR SEQ ID NO: 47 P 61 IKSKILIPXSAP SEQ ID NO: 48 P 62TNVSTQNIVQPL SEQ ID NO: 49Peptide sequences were synthesized and the ability of the peptides toprotect against polyurethane oxidation was determined.

Example 11 Selective Binding of a Peptide Selected to Target Baked-onEgg Soil on Stainless Steel or Glass

Egg soil was prepared by using the yolks from fresh eggs. The yolks wererinsed in cold water, then forced through a strainer into a beaker. Thebeaker was placed into a 140° F. water bath, and the egg yolk cooked for30 minutes with constant stirring. After 30 minutes, the beaker wasplaced into an ice bath to cool the yolks to room temperature withconstant stirring. #316 Stainless steel foil disks were cut to adiameter of 7/32″ using a die with an expulsion to fit an NAEF punchpress (MS Instrument Company, Stony Creek, N.Y.). These were used asboth the substrate for the target, baked-on egg soil, and theanti-target, unsoiled disks. Before use, the disks were washed in milddetergent, and rinsed thoroughly in deionized water.

Egg soiled 316 stainless steel disks and egg soiled glass beads wereplaced into a Costar 96-well flat bottom plate. For each peptidelibrary, three clean stainless steel (SS) disks or glass beads(anti-targets) were placed into adjacent wells in a 96 well plate. Anegg soiled (target) disk or bead was placed in the adjacent well. Intothe first well (A) containing a clean disk or bead was added 150 μLdetergent and 10 μL phage library—C7C, linear 7-mer, or wild type phage.The samples were incubated at room temp for 20 min with gentle agitationand the supernatant containing unbound phage peptides was transferred tothe next well and the process repeated a total of three times. Thesupernatant was then transferred to the egg soiled coupon or bead andincubated for 30 minutes with gentle mixing. The samples were thentransferred to a fresh well and washed a total of 38 times as follows:3× in 200 μL detergent solution, 3.5 g/l powder automatic dishwashingdetergent, 30× in 250 μL PBST, and 5× in 200 μL PBS.

The washed disks or glass beads were transferred to a 0.5 mL PCR tube.The PCR reaction was run directly on the egg soiled disks or beads using200 μL of reaction mixture using the Qiagen HotStart® kit and 50 μL ofmineral oil. PCR amplification was performed using 1 cycle at 95° C. for15 min to initiate the reaction, followed by 30 cycles of denaturationat 94° C. for 30 sec, annealing at 58° C. for 30 sec, and extension at72° C. for 30 sec, and concluding with 1 cycle for 10 min at 72° C. forelongation. The 278 bp product as analyzed on an 2% agarose gel alongwith molecular weight markers. As shown in FIG. 8 for stainless steel asthe anti-target, a PCR product is visible for the linear 7-mer library,and there was no visible signal for wild-type (WT) phage control. Asecond PCR amplification was conducted and the PCR product was clonedinto a TOPO® TA vector (Invitrogen) for sequencing as summarized inTable 7.

TABLE 7  Amino Acid sequences that bind to egg-soil onstainless steel and not to stainless steel Amino Acid Clone ID SequenceFrequency^(a) E 1  LSPHLAR 4 SEQ ID NO: 50 E 2  THRPDWD 3 SEQ ID NO: 51E 3  APKSFKT 2 SEQ ID NO: 52 E 4  AYSQWKY 2 SEQ ID NO: 53 E 5  DFSPQLD 2SEQ ID NO: 54 E 6  GLFEWRV 2 SEQ ID NO: 55 E 7  ILNHPPN 2 SEQ ID NO: 56E 8  LNQKNVT 2 SEQ ID NO: 57 E 9  LPSEFLR 2 SEQ ID NO: 58 E 10 MPGATSL 2SEQ ID NO: 59 E 11 QMSAQWR 2 SEQ ID NO: 60 E 12 SNTAIWR 2 SEQ ID NO: 61E 13 TASPMPL 2 SEQ ID NO: 62 E 14 VALPTLT 2 SEQ ID NO: 63 ^(a)Number ofmultiple times this amino acid sequence occurred out of 118 clonesThe sequences were cloned into a subtilisin protease gene and theaffinity for egg soil was determined in a proteolytic assay.

Example 12 Specific and Selective Binding of a Selected Peptide toTarget Tea Stains on Ceramic

Using the methods described in Example 1, peptides that bind to tea onceramic in the presence of automatic dishwashing detergent wereidentified after two rounds of selective targeting. The target boundpeptide sequences are summarized in Table 8.

TABLE 8  Amino Acid sequences that bind to tea on ceramic Clone IDAmino Acid Sequence T 1  LDYKHDL SEQ ID NO: 64 T 2  SAAADYLSEQ ID NO: 65 T 3  TPGPLFL SEQ ID NO: 66 T 4  DXQDNIW SEQ ID NO: 67 T 5 MPQPSSM SEQ ID NO: 68 T 6  LTITIQE SEQ ID NO: 69 T 7  XPGPLFLSEQ ID NO: 70 T 8  TNFATXL SEQ ID NO: 71 T 9  DARNALF SEQ ID NO: 72 T 10WTSLISN SEQ ID NO: 73 T 11 ACWLRPXLHC SEQ ID NO: 74 T 12 NLSSSNKHAVGNSEQ ID NO: 75 T 13 YVHRPNA SEQ ID NO: 76 T 14 GSYDPKEFHHPQ SEQ ID NO: 77

Example 13 Screening for Peptides Selected to Target Human Skin and notHair

Two 3 inch strands of dark human hair (International Hair Importers &Products, White Plains, N.Y.) were placed in BSA blocked 50 ml conicaltubes containing 10 ml of a 2% Neutrogena® body wash (Neutrogena Corp.)solution in DI water. 10 μL of cyclic 7-mer or linear 12-mer peptidelibraries (10¹⁰ pfu/μl), or wild type phage (10⁹ pfu/μl) were added andthe samples mixed at room temperature for 15 min with rotatory shaking(30 rpm). The unbound supernatant was transferred to a new tubecontaining an additional two 3 inch strands of dark hair, and incubatedat room temperature for 15 min with rotary shaking. After this secondhair incubation, 500 μl of the solution was transferred to the surfaceof human skin tissues (EpiDerm™, MatTek Corp. Ashland, Mass.) in a 6well culture plate containing 0.9 mL tissue culture media (MatTek Corp)for 30 minutes at room temperature with gentle agitation. The skintissues were removed and washed 2× in 50 mls of 2% body wash for 5 mineach and 3× in 50 mls of PBS for 5 min each in blocked 50 mL conicaltubes. After the final PBS wash, the skin tissues were frozen at −20° C.followed by PCR of the target bound ligand phage.

Example 14 Screening for Peptides Selected to Target Human Hair and NotSkin

Pre-equilibrated skin tissues were placed into a 6 well culture platecontaining fresh 0.9 mL tissue culture media and 300 μl of a 2%Neutrogena® body wash containing, 10 μL of cyclic 7-mer or linear 12-merpeptide libraries (10¹⁰ pfu/μl), or wild type phage (10⁹ pfu/μl) wereadded to the skin surface. The samples were incubated at roomtemperature for 15 min with gentle agitation. The unbound supernatantwas transferred to a new well containing skin tissue and the procedurewas repeated. The incubation solution was transferred to nine 3 inchdark hair (International Hair Importers & Products, White Plains, N.Y.)strands in 50 ml tubes containing 10 ml of 2% body wash for 30 minutesat room temperature with rotatory shaking (30 rpm). The hair sampleswere then washed with 1×50 mls, 2×50 mls, or 4×50 mls of 2% body wash;Wash cycles in PBS followed (1×25 mls for 5 min, 1×25 mls for 2 min,2×50 mls for 5 min each, 150 mls total). After the final PBS wash thehair samples containing bound phage peptides were frozen at −20° C. PCRamplification of target-bound phage was carried out as described inExample 1 with minor modifications. PCR reactions contained 50 μg of BSAto prevent inhibition of the PCR reactions by hair or skin.

Example 15 ELISA Assay for Selective Binding of Peptides that TargetHuman Hair and Not Skin or Target Skin and Not Hair

Peptide sequences identified in Examples 13 and 14 along with a randomcontrol peptide were C-terminally labeled with the sequence GGGK(biotin). The sequence LESTPKMK (SEQ ID NO: 115) contains the consensussequence LEST and was isolated on hair. FTQSLPR (SEQ ID NO: 116)contains the consensus sequence TQSL and was isolated on skin. YGGFMTSE(SEQ ID NO: 117) is a control peptide.

Dark brown hair (3″ long, 4 each), moistened with 2% body wash andpre-equilibrated human skin tissues, were placed in the wells of a 24well plate. 1 ml of a 200 μM solution of the biotinylated peptide in 2%Neutrogena body wash was added to the hair and skin samples andincubated 30 min at room temperature with gentle agitation. The solutionwas then pipetted off and the hair and skin samples transferred withclean tweezers to a 50 ml conical tube, washed once with 50 ml of 2%body wash, twice with 50 ml of water, and once with 50 ml of PBS; eachwash step took 5 min and was performed on a rotary shaker at 20 rpm. Thehair and skin samples were then transferred with clean tweezers to afresh 24 well plate where 1 ml of streptavidin conjugated horseradishperoxidase (diluted 1/1000 in PBS) was added for 1 hr at roomtemperature under gentle rocking. Excess streptavidin HRP was removed bywashing twice with 50 ml of PBS (5 min, 20 rpm each) in a 50 mL conicaltube. The hair and skin samples were transferred to fresh wells and 1 mlof H₂O₂/OPD solution was added and the color left to develop at roomtemperature. FIG. 9 shows that peptide binding is selective for therespective targets, relative to the control peptide.

What is claimed is:
 1. A peptide comprising the amino acid sequence ofany one sequence of SEQ ID NOs:18-26, 51-63, 64, 66-68, 71, 73-77,29-49, and 103-113.